Peptide or protein containing a C &#39;-D loop of the CD28 receptor family

ABSTRACT

The invention relates to a protein or peptide comprising the C′-D loop of a member of the CD28 family, uses thereof and mAbs obtainable therefrom.

FIELD OF THE INVENTION

[0001] The invention relates to a protein or peptide containing a partial sequence of a member of the CD28 receptor family, a nucleic acid coding for such a peptide, a plasmid containing such a nucleic acid, hybridoma cells forming monoclonal antibodies (mAbs) binding to such a peptide, mAbs obtainable from such hybridoma cells, and methods of use of the peptide and the mAbs.

DEFINITIONS

[0002] Monoclonal antibodies are antibodies being produced by hybrid cell lines (so-called hybridomas) typically resulting from the fusion of a B cell of animal or human origin producing antibodies with a suitable myeloma tumor cell.

[0003] The amino acid sequence of human CD28 is known under the accession no. NM_(—)006139.

[0004] The amino acid sequence of human CTLA-4 is known under the accession no. L15006.

[0005] The amino acid sequence of human ICOS is known under the accession no. AJ277832.

[0006] The amino acid sequence of human PD-1 is known under the accession no. U64863.

[0007] The C′-D loop of CD28 comprises the amino acids 52 to 66 of the above CD28 sequence (numbering according to FIG. 7, see also Ostrov, D. A. et al.; Science (2000), 290:816-819). The term C′-D loop will in the following also comprise any partial sequences thereof.

[0008] A loop or a binding site arranged therein is freely accessible, if there is for a defined binding partner no steric hindrance for the binding site in the loop by the sequences or molecules outside of the loop.

[0009] Activation of T lymphocytes is the increase of metabolic activity, increase of the cell volume, synthesis of immunologically important molecules and initiation of the cell division (proliferation) of T lymphocytes as a response to an external stimulation. Inhibition is the opposite process. For example are such processes caused by occupation of the CD28 molecule on T cells by special CD28-specific monoclonal antibodies. The activation of the T lymphocytes with the described side effects is part of the physiologic immune reaction, in pathologic situations however there may be lost control thereof (lympho-proliferative diseases), or may be insufficient (immunodeficiency).

[0010] Modulation of the proliferation of T cells is either the increase of the activity (for a pathologically insufficient activation) or reduction or inhibition of the activity (for pathologically lympho-proliferative diseases).

[0011] Several sub-groups of the T cells means at least sub-groups of CD4 and CD8 T cells expressing CD 28.

[0012] An analogous peptide is a peptide the amino acid sequence of which differs from that one of the peptide to which it is analogous, which binds however a defined binding partner with at least the same affinity. Deviations in the sequence may be deletions, substitutions, insertions and elongations. An analogous peptide will usually comprise a tertiary (partial) structure and/or exposition being very similar to the peptide, in a (cell surface) protein, and otherwise only needs to comprise or form a binding site for the defined binding partner in the section analogous to the immediate binding section of the peptide.

[0013] A mimicry compound of a mAb is a natural or synthetic chemical structure behaving in a binding assay as a defined mAb mimicrying the mimicry compound.

[0014] A mimicry compound of a C′-D loop is a natural or synthetic chemical structure to which specifically bind mAbs being superagonistic and specific for a member of the CD28 family.

[0015] The term mAbs comprises, in addition to structures of the usual Fab/Fc constructions, also structures consisting of or comprising the Fab fragment only. It is also possible to exclusively use the variable region, the fragment of the heavy chains being connected in a suitable manner, for instance also by means of synthetic bridge molecules, with the fragment of the light chain, in such a way that the binding regions of the chains form the antibody binding site. The term antibody also comprises (complete) chimeric and humanized antibodies.

[0016] Superagonistic modulation of the proliferation of T cells means that no costimulation, i.e. no further binding event in addition to a binding of a mAb or of a mimicry compound to a member of the CD28 family is required for the stimulation or inhibition of the proliferation.

[0017] A screening method comprises the use of a target, for instance a partial sequence from CD28, one or more known or unknown substances being contacted with the target and a binding event being detected or not detected. In the case of the detection of a binding event, the substance is selected. In the case of the use of a mixture of substances, typically a deconvolution follows a selection of the mixture for the purpose of the determination of binding components in the selected mixture.

[0018] As a CD28 family is designated a group of T cell surface receptors having an immuno-regulatory activity. This may be either stimulating, as in the case of the CD28, or inhibiting, as in the case of the CTLA-4. To the CD28 family belong CD28, CTLA-4, PD-1 and ICOS.

[0019] A substrate can be soluble, insoluble and/or immobilized. A substrate can be formed of any natural or synthetic molecules, for instance of amino acid chains, among others. In this respect a protein or a peptide in the terminology of this text needs not necessarily be a protein or a peptide according to the conventional definition. Usually, however, a protein or peptide according to the terminology used herein is also a protein or peptide in the usual terminology.

BACKGROUND OF THE INVENTION AND PRIOR ART

[0020] For the understanding of the invention, first the following technological background is important. The activation of resting T cells for the proliferation and functional differentiation first requires the occupation of two surface structures, so-called receptors: i.e. of the antigen receptor having a different specificity from cell to cell and being necessary for the detection of antigens, for instance viral fission products; and of the CD28 molecule expressed in an identical manner on all resting T cells with the exception of one sub-group of the human CD8 T cells, said CD28 molecule binding in to ligands on the surface of other cells. This is called the costimulation of the antigen-specific immune reaction by CD28. In cell culture, these processes can be simulated by occupying the antigen receptor and the CD28 molecule by suitable mAbs. In the classical system of the costimulation, neither the occupation of the antigen receptor nor of the CD28 molecule alone will lead to the T cell proliferation, the occupation of both receptors is however effective. This observation has been made for T cells of man, mouse and rat.

[0021] There are known, however, also mAbs that can alone initiate the T cell proliferation. Such a superagonistic (that is independent from the occupation of the antigen receptor) activation of resting T cells has been observed in the following systems: in the document Brinkmann et al., J. Immunology, 1996, 156: 4100-4106, it has been shown that a very small fraction (5%) of human T lymphocytes carrying the surface marker CD45 RO being typical for resting T lymphocytes, is activated by the CD28-specific mAb 9.3 normally requiring costimulation with addition of the growth factor interleukin-2 (IL-2) without occupation of the antigen receptor. In the document of Siefken et al., Cellular Immunology, 1997, 176: 59-65, it has been shown that a CD28-specific mAb produced in a conventional manner, i.e. by immunization of mice with human cells, can activate in cell culture a sub-group of human T cells without occupation of the antigen receptor for the proliferation, if CD28 is occupied by this mAb and the cell-bound mAbs are in addition cross-linked with each other by further antibodies. It is common to the in this respect known antibodies that only a small fraction of the T cells can be activated.

[0022] In the document of Tacke et al., Eur. J. Immulog., 1997, 17: 239-247, two kinds of CD28-specific monoclonal antibodies with different functional properties have been described: costimulatory mAbs costimulating the activation of resting T cells with a simultaneous occupation of the antigen receptor only; and superagonistic mAbs being able to activate T lymphocytes of all classes in vitro and in animal experients for the proliferation, without occupation of the antigen receptor. Both in this respect known mAbs originate from an immunization with cells, on the surface of which rat CD28 is expressed, and which are obtainable by different selections directed to their respective properties. Finally, from WO 98/54225 is known another superagonistic mAb, namely CMY-2.

[0023] The in this respect known superagonistic mAbs do not meet in their stimulatory effect the requirements with regard to the strength of the activating effect or the width of the activated sub-populations of T lymphocytes or do not have the required human specificity.

TECHNICAL OBJECT OF THE INVENTION

[0024] The invention is based therefore on the technical object to provide means, by use of which superagonistic compounds can be found which bind to one or several members of the CD28 family and have an improved stimulatory or inhibiting effect, as well as to specify such compounds.

[0025] The Findings the Invention is Based On.

[0026] The invention is based on the examination of the binding regions of superagonistic mAbs at CD28 as well as the interaction found in these experiments of the C′-D loop of CD28 with superagonistic mAbs. Further, the invention is based on the finding that a corresponding binding region for superagonistic mAbs can be found in the other members of the CD28 family, namely there, too, the C′-D loops. From this basic findings, various aspects for technical teachings of the invention can be deducted.

[0027] Basics of the Invention and Preferred Embodiments.

[0028] For achieving the technical object, the invention teaches a protein or peptide comprising the C′-D loop of a member of the CD28 family, or comprising a peptide being analogous thereto or comprising a mimicry compound thereto, not however a member of the CD28 family. In other words, the essential element of a protein or peptide according to the invention is the C′-D structure (or of an analogous/mimicry substance thereto), and that irrespective of whether and which sequences follow on both sides of the loop. It is only essential that the loop structure is sufficiently exposed, in order to offer access for superagonistic mAbs or mimicry compounds and to prevent in the case of the specific binding possibility a binding not for steric reasons. According to the invention, it is surprising that all found superagonistic CD28-specific mAbs bind to the C′-D loop, whereas the not superagonistic CD28-specific mAbs do not bind thereto. By a single group of target structures with regard to their spatial distribution on members of the CD28 family, thus prospective effective substances against a number of different diseases are made accessible.

[0029] In the embodiment of a peptide, in particular of an oligopeptide (4 to 9 amino acids) or a polypeptide (10-100 amino acids) or of a mimicry compound thereto, it is preferred that the ends thereof are each bound to a binding position of a substrate, the binding positions of the substrate being spatially arranged with regard to each other according to the binding positions for the C′-D loop, the C′-D loop or the peptide being analogous thereto or the mimicry compound thereto being fixed in a three-dimensional configuration according to the C′-D loop, the bound C′-D loop or the peptide being analogous thereto or the mimicry compound thereto being freely accessible for antibodies or mimicry compounds thereto, and the substrate not being a member of the CD28 family without a C′-D loop. Thus a three-dimensional structure permitting a binding with superagonistic substances is provided.

[0030] In detail, a peptide or protein according to the invention may comprise an amino acid sequence seq.-ID 41 (human CD28 loop), not however be human CD28, seq.-ID 42 (human CTLA-4 loop), not however be human CTLA-4, seq.-ID 43 (human ICOS loop), not however be human ICOS, or seq.-ID 44 (human PD-1 loop), not however be human PD-1. One or two amino acids may be added according to FIG. 7 to the 3′ end and/or the 5′ end. But partial sequences thereof may also be comprised in the peptides according to the invention, for instance according to the sequences seq.-ID 1 to 4, respectively. The seq.-ID 5 to 10 indicate variants of the human CD28 loop. The seq.-ID 12 to 17 indicate variants of the human CTLA-4 loop. The seq.-ID 19 to 24 indicate variants of the human ICOS loop. The seq.-ID 26 to 31 indicate variants of the human PD-1 loop. One or more amino acids of the sequence 11 may be added according to FIG. 7a to one of the sequences 1, 7 or 9. One or more amino acids of the sequence 18 may be added according to FIG. 7a to one of the sequences 2, 14 or 16. One or more amino acids of the sequence 25 may be added according to FIG. 7a to one of the sequences 3, 21 or 23. One or more amino acids of the sequence 32 may be added according to FIG. 7a to one of the sequences 4, 28 or 30. The above sequences are sections according to the invention, to which superagonistic mAbs will specifically bind. It can in particular be seen, when comparing the sequences, that the primary structure of the loop is specific for the respective family members. By selection of the C′-D loop of a specific member and thus by application of substances having specificity for this selected loop, thus alternatively an activation or an inhibition of the proliferation can be obtained.

[0031] A (CD28-specific) protein or peptide according to the invention or a mimicry compound thereto can be identified by that one or more prospective proteins, peptides or mimicry compounds are subjected to a binding test with e.g. one of the mAbs 9D7 or 5.11A, and binding peptides are selected. The mentioned mAbs are new superagonistic CD28-specific mAbs, which are described in detail in the experimental section hereof. By means of corresponding mAbs with specificity for a C′-D loop of another CD28 family member, proteins, peptides or mimicry compounds according to the invention and being specific for the other members can be identified. Such corresponding mAbs may be obtained in an analogous manner.

[0032] The invention further relates to a nucleic acid coding for a peptide according to the invention or for a protein comprising such a peptide, not however coding for a member of the CD28 family, and to a vector, e.g. plasmid, comprising such a nucleic acid, operably linked to a suitable promotor.

[0033] The peptide, protein according to the invention or a mimicry compound thereto according to the invention can be used in a method for producing mAbs which superagonistically modulate the proliferation of T cells of several to all sub-groups, a non-human mammal being immunized with the protein or peptide or the mimicry compound thereto, from the non-human mammal cells being taken, hybridoma cells being produced from the cells, and such obtained hybridoma cells being selected, the culture supernatant of which contains mAbs, which bind to the C′-D loop of the protein or peptide or the mimicry compound thereto, such hybridoma cells and mAbs obtainable with such hybridoma cells. Human mAbs according to the invention can alternatively however also be produced by that B lymphocytes are selected which bind to the loop, and that their expressed immunoglobulin genes are cloned. Furthermore, human mAbs can be isolated from phage libraries. The average man skilled in the art is without any problems in a position, using his knowledge, to execute such alternative methods, so that no detailed description is needed here.

[0034] By using the invention, new superagonistic CD28 family specific mAbs and/or mimicry compounds can however also be identified. Therefore the invention also relates to the use of a peptide, of a protein according to the invention or of a mimicry compound thereto according to the invention in a screening method for the identification of substances superagonistically modulating the proliferation of T cells of several to all sub-groups, a prospective substance or a mixture of prospective substances being subjected to a binding assay with the peptide or protein or mimikry compound thereto, and substances binding to the peptide or protein or mimikry compound thereto being selected. In principle, any conventional binding assay can be used. Of special importance may be here the search for mimicry compounds, since these are typically so-called small molecules having pharmacological advantages over macromolecules. In such a screening method for mimicry compounds, a substance library can be screened with high throughput. Both aforementioned uses may be carried out with a native CD 28 receptor family member as well.

[0035] A peptide, protein or mimicry compound thereto according to the invention as well as the mAbs or mimicry compounds thereto according to the invention have therapeutic relevance, since thereby lymphoproliferative diseases may be treated by inhibition of the proliferation, as well as immunodeficiency diseases by activation of the proliferation. The induction of effector functions, e.g. secretion of effector substances, is also possible. This is achieved by selection or design of the mAb or of the mimicry compound according to a specificity and high affinity for a specific member of the CD28 family. If a higher specificity/affinity is desirable, for instance, for controlling surprising side effects, the process may be such that a second ligand in addition to the mAb or the mimicry compound with specificity for the special family member is searched, and the second ligand is linked, after an analysis of the relative spatial positions of the bound two ligands with respect to each other, by a bridging molecule with the mAb or the mimicry compound. The determination of the position of two ligands with respect to each other after binding to a target can for instance be made by X-ray structure analysis or multi-dimensional NMR correlation spectroscopy, for instance ¹⁵N/¹H NMR. A second ligand can be determined by conventional screening methods, the special CD28 family member being used as a target. It is understood that the second ligand does not bind at the C′-D loop, but spaced thereto. On the other hand it is possible that a peptide, protein or mimicry compound according to the invention thereto hot having an otherwise physiological effect, competitively binds natural ligands of the members of the CD28 family, and thus creates a reverse effect by prevention of a pathologically caused natural activation or inhibition.

[0036] Therefore the invention relates on one hand also to the use of a peptide, protein or mimicry compound thereto according to the invention for producing a pharmaceutical composition for the modulation of the physiological T cell proliferation, wherein a such compound is optionally mixed with suitable carrier and auxiliary compounds and galenically prepared for the desired mode of administration, e.g. injection i.v or i.p.

[0037] On the other hand, therefore, the invention relates to the use of a mAb according to the invention or a mimicry compound thereto according to the invention for producing a pharmaceutical composition for the treatment of diseases with pathologically reduced CD4 T cell numbers, in particular AIDS, for producing a pharmaceutical composition for the treatment following stem cell transplantations after chemo or radio therapy of leukemic diseases, for producing a pharmaceutical composition for multiplying and/or qualitatively influencing immune reactions after vaccinations, and for producing a pharmaceutical composition for the treatment of autoimmune-inflammatory diseases. Mixing and galenic preparation is performed in an according manner.

[0038] The invention finally relates to therapeutic methods, wherein to a person suffering from a proliferative or immunodeficient disease, a pharmaceutical composition according to the invention is administered.

[0039] In the following, the invention is described in more detail with respect to examples representing embodiments only. There are:

[0040]FIG. 1: stimulation of T lymphocytes from the rat with various CD28-specific mAbs (a: costimulation, b: superagonistic, i.e. direct stimulation),

[0041]FIG. 2: a sequence comparison between mouse, rat and human CD28 in the section of the C′-D loop (in box),

[0042]FIG. 3: experimental results for the localization of the binding site of superagonistic mAbs at the CD28 molecule of the rat,

[0043]FIG. 4: binding of various human CD28-specific mAbs to CD28 (a) and costimulatory (b) and superagonistic, i.e. directly stimulatory (c) activity of the mAbs of FIG. 4a,

[0044]FIG. 5: binding tests that show that human CD28-specific superagonistic mAbs specifically bind to the C′-D loop,

[0045]FIG. 6: a three-dimensional representation of CD28 with marking of the C′-D loop,

[0046]FIG. 7: sequence alignment under emphasis of the C′-D loop or analogous structures, for various members of the CD28 family,

[0047]FIG. 8: experiments for the activation of cells by means of human CD28-specific mAbs and mutated mouse CD28 molecules,

[0048]FIG. 9: representation of the sequences seq.-ID 33-40 (a-h), and

[0049]FIG. 10: humanized variable domain of the mAb 5.11A (light chain: VLC5.11=a; heavy chain: VHC5.11=b)

[0050]FIG. 1 shows the stimulation of freshly isolated T lymphocytes from the rat in the form of a 3H thymidine incorporation. The approach corresponds to the one described in document WO98/54225, to which reference to a full extent is made here and in the following, and the scope of disclosure of which is hereby incorporated in the present text. In FIG. 1a is shown the costimulation, i.e. in all wells, T cell receptors (TCR) specific mAbs were bound to the plastic surface. For lack of costimulation, the negative control (uppermost row) does not show any incorporation. Costimulation is then given by the addition of CD28-specific mAbs in a soluble form. All the different CD28-specific mAbs used are shown. This series of various CD28-specific mAbs originate from an approach of the immunization and production of hybridoma cell lines described in WO98/54225 already. These are culture supernatants containing sufficient CD28-specific mAbs for a saturating binding to the 2×10 cultivated cells. From FIG. 1a can be taken that all of these mAbs are able to activate in a costimulating manner, i.e. to induce in presence of the anti-TCR mAbs the thymidine incorporation. In FIG. 1b is shown the stimulation in absence of TCR specific mAbs. This experiment has also been performed as described in document WO98/54225. It can be seen that only two mAbs are capable to stimulate the T lymphocytes in absence of a TCR signal. These mAbs have thus superagonistic activity.

[0051] Furthermore it has been examined whether costimulatory and superagonistic CD28-specific mAbs bind to different sections of the CD28 molecule. The mAbs were produced by immunization of mice with CD28 from the rat; as expected, they all do not react with mouse CD28 (not shown). Since the mAbs can thus only recognize such sections of the rat CD28 molecule which differ from that of the mouse, first a sequence comparison between CD28 from the mouse and the rat was performed (see FIG. 2, upper part). The differences between the two species are highlighted. For the designation of the amino acids, the one-letter code was used. As prototypes for a conventional rat CD28-specific mAb was used JJ319, for a superagonistic mAb JJ316 (see WO98/54225).

[0052] In FIG. 3, the mapping of the binding region is shown. Expression plasmids were constructed, wherein one part of the extracellular domain of CD28 originates from the mouse, another one from the rat. This is shown by bars or lines, respectively; on the right-hand side thereof the binding of the mAbs JJ316 and JJ319 to mouse fibroblasts (L929 cells) is represented, which have been transfected with these expression plasmids. In the first two lines of FIG. 3 (m/r and r/m 1-37), the binding of both antibodies to the “right-hand” half of the sequence is mapped: Both will bind if this originates from the rat. In the reverse construct (rm CD28 1-37, left-hand rat, right-hand mouse), there is no binding. In the third line (m/r CD28 1-66) it is shown that JJ316 does not bind anymore, whereas the still present part of the rat sequence (“right-hand”) is still sufficient for a detection by JJ319. Thus, the two mAbs detect different epitopes on the CD28 molecule, and the binding of the superagonist JJ316 is located in that region which originated in the construct of the first line, not however in the construct of the third line from the rat. A clear candidate therefor is the section in the box of FIG. 2.

[0053] In lines 4 and 5 of FIG. 3, therefore first two and then three amino acids were so modified in this region of the mouse CD28 molecule that they will now represent the rat sequence. By this “transplantation” of three amino acids only, the binding capability for mAb JJ316, not however that of JJ319 could be transferred. In Table 1 the binding data for the whole group of CD28-specific mAbs are summarized. There exists a unique correlation: a significant binding to the C′-D loop of the rat which has been created by transfer of the amino acid positions 62, 64 and 65 to the CD28 molecule of the mouse, could be found for the two superagonistic mAbs JJ316 and 5S38 17 only, not however for the conventional (only costimulatory) mAbs. A costimulatory mAb (5S35) detects the epitope in the box to a very weak degree and binds very strongly to the “conventional” epitope.

[0054] The next figures deal with superagonistic human-specific mAb. These have also been produced in mice, thus do not react with the CD28 molecule of the mouse. The mice were immunized with human CD28-transfected A20/J mouse B lymphoma cells (see WO98/54225) and additionally boostered prior to the fusion with commercially available human CD28-Fc fusion protein (bought from R and D Systems) . In a series of fusion experiments, 24 out of several thousand cell lines were identified which produce human CD28-specific mAbs (binding to mouse L929 cells expressing human CD28, not however to untransfected L929 cells), in an analogous manner to the screen in document WO98/54225. From these, two showed the desired superagonistic activity (9D7 and 5.11A), whereas all new mAbs have a conventional costimulatory activity. In the following, in particular these two superagonistic mAbs will be described. As an example for a conventional human CD28-specific mAb, the also newly generated mAb 7.3B6 is used.

[0055]FIG. 4a shows that the used preparations of the three new mAbs bind comparatively well and also with comparable titer to human T lymphocytes. An experiment is shown, wherein freshly isolated mononuclear cells from the human blood (so-called PBMC) were first treated with different dilution steps of the used mAbs on ice; then they were washed, and the bound mAb was made visible by a secondary antibody marked by a fluorescence dye, said secondary antibody specifically detecting the bound mouse mAbs. By using another mAb detecting human CD4 T cells, and to which a second fluorescence dye was bound, the binding of the titrated mAbs could be determined by electronic gating selectively for the CD4 T lymphocytes. By “MFI” is given the average fluorescence intensity being a measure for the amount of the bound CD28-specific mAb. The concentrations represent 3-fold dilutions of a standardized original preparation. It is fully normal that in this test the highest concentration gives a weaker signal than the following titration steps; this has to do with the avidity (bivalent binding) of mAbs and does not play any role in the context discussed here.

[0056] In FIG. 4b and c the capability of these three new CD28-specific mAbs to stimulate—in presence and absence of a TCR signal—freshly isolated human T cells to growth is compared. Again, a 3H thymidine incorporation is shown, as described above for the rat. For FIG. 4b, the wells were coated with a mAb which reacts with the human TCR/CD3 complex. Thus, the costimulation was measured. It can be seen that the proliferation without costimulation with one the mAbs fails to appear (negative control), all three antibodies are however capable to stimulate the cell division. For FIG. 4c, the absence of a TCR/CD3-specific mAb was selected. Only the antibodies 9D7 and 5.11A could stimulate in a superagonistic way.

[0057] After the epitope for superagonistic mAb for the rat has been defined, and two new superagonistic mAbs having specificity for human CD28 have been isolated, it has been checked whether these mAbs bind to the corresponding position of the human CD28 molecule. As can be seen from FIG. 2, the CD28 molecules of mouse and man differ in numerous positions. Based on the mapping of the superagonistic epitope for the rat, it has therefore been directly checked whether the binding site for the superagonistic epitope on human CD28 to the CD28 molecule of the mouse can be achieved by “transplantation” of the five amino acids of this homologous region. The results are shown in FIG. 5. Taking into account the background of the homogeneously represented mouse sequence for the extracellular domain of the CD28 molecule (center) the exchanged (mouse to man) amino acid positions are shown as lines (bottom). The numbers at the side indicate in addition the individual positions and mutations (F60V means for instance that at position 60 the phenylalanine of the mouse has been replaced by a valine of the human sequence). Adjacent thereto, the binding of the three examined mAbs is represented. As the figure shows, all three mAbs detect human CD28, only the two mAbs 9D7 and 5.11A however react with the mouse molecule, into which the five amino acids of the human CD28 have been transplanted at the crucial position. Taking into account the great variety of differences, this specific generation of the reactivity is surprising and confirms to a full extent the findings, derived from the experiments with rat CD28, that superagonistic mAbs must bind to a specific, namely exactly this site of the molecule.

[0058] In FIG. 6 is shown a three-dimensional model of the CD28 molecule. The newly identified binding region is highlighted. It corresponds to the sequence in the box in FIG. 2. Concerning its structure, the extracellular domain of CD28 belongs to the so-called immunoglobulin super family being characterized by two superimposed 8 sheets as a basic structure. The labeling of these bands follows a pattern as given in the literature. It is important for the representation shown here that the region identified as an epitope for superagonistic CD28-specific mAbs in rat and mouse is described by “C′-D loop”. It has thus been shown that mAbs having specificity for the C′-D loop of the CD28 molecule show superagonistic activity, thus can be used for the activation of T lymphocytes in the meaning of the document WO98/54225. The superagonistic activity of C′-D loop-specific mAbs in rat and man shows that therein not the sequence of the epitope, but its position or shape is important.

[0059] CD28 belongs to a family of cell surface receptors with immuno-regulatory activity. This is either stimulating (CD28, ICOS) or inhibiting (CTLA-4, PD-1). In FIG. 7, the sequences of the known members of the CD28 family are shown in the sense of an “alignment”. The C′-D loop for CD28 is highlighted. Analogous loops of the other molecules (in box) are a correspondingly favorable target structure for the development of superagonistic ligands. It should be noted, with regard to FIG. 7, that “−” is a gap in the alignment, i.e. the amino acids following thereto are immediately connected to each other.

[0060] In the experiments of FIG. 8, it has been examined whether mAbs according to the invention having specificity for the C′-D loop of the rat or man do not only bind to the mouse CD28 with “transplanted” C′-D loop of the rat or man (see FIGS. 3 and 5), but whether there is really an activation. For this purpose, T tumor cells of the mouse, BW, were transfected either with the construct of FIG. 3, line 5 (rat C′-D loop transfer) or with the construct of FIG. 5, line 3 (human C′-D loop). The activation of these cells is not measured by cell division (they proliferate anyway), but by the production of the cytokine IL-2. FIG. 8 shows first that without stimulation there is no IL-2 production (negative control). The stimulation with a T cell receptor-specific mAb induces IL-2 production (positive control). FIG. 8a shows the results when using the superagonistic mAb JJ316 of the rat, whereas FIG. 8b shows the results for the human C′-D loop-specific mAb 5.11A. In either case the respective cell lines are stimulated for the IL-2 production. As expected, there is however no stimulation by “conventional” CD28-specific mAbs, since these do not only not bind to the C′-D loop, but cannot detect the construct at all, because they are specific for rat or human-specific sequences not contained in the construct.

[0061] In FIG. 9a is shown the nucleic acid sequence of the variable region of the light chain of a mAb 9D7 according to the invention (seq.-ID 33). FIG. 9b shows the peptide coded thereby (seq.-ID 34). FIG. 9c shows the nucleic acid sequence of the variable region of the heavy chain of this mAb (seq.-ID 35). FIG. 9d is the peptide coded thereby (seq.-ID 36).

[0062] In FIG. 9e is represented the nucleic acid sequence of the variable region of the heavy chain of a mAb 5.11A according to the invention (seq.-ID 37). FIG. 9f shows the peptide coded thereby (seq.-ID 38). FIG. 9g shows the nucleic acid sequence of the variable region of the light chain of this mAb (seq.-ID 39). FIG. 9h shows the peptide coded thereby (seq.-ID 40). FIG. 10 shows the humanized variable domain of the mAb 5.11A. FIG. 10a is the light chain, and FIG. 10b the heavy chain. TABLE I Binding of anti-rat CD28 mAbs to mouse and rat CD28 and various CD28 mutants mCD28, S62P m/rCD28 mAb Mouse CD28 Rat CD28 A64V, EG5G Mva11269I Control — — — — JJ316 — + + + + + + — JJ319 — + + + — + + + 5S28 — + + — + + + 5S38.17 — + + + + + + — 5S247 — + + + — + + + 5G40/3 — + + + — + + + 5G87 — + + — + + 5G111 — + + — + + 5G35 — + + + + + + +

[0063]

1 44 1 6 PRT Artificial Novel Sequence 1 Val Tyr Ser Lys Thr Gly 1 5 2 5 PRT Artificial Novel Sequence 2 Phe Leu Asp Asp Ser 1 5 3 6 PRT Artificial Novel Sequence 3 Val Ser Ile Lys Ser Leu 1 5 4 6 PRT Artificial Novel Sequence 4 Gln Pro Gly Gln Asp Cys 1 5 5 4 PRT Artificial Novel Sequence 5 Tyr Ser Lys Thr 1 6 5 PRT Artificial Novel Sequence 6 Tyr Ser Lys Thr Gly 1 5 7 5 PRT Artificial Novel Sequence 7 Val Tyr Ser Lys Thr 1 5 8 6 PRT Artificial Novel Sequence 8 Tyr Ser Lys Thr Gly Phe 1 5 9 7 PRT Artificial Novel Sequence 9 Val Tyr Ser Lys Thr Gly Phe 1 5 10 5 PRT Artificial Novel Sequence 10 Ser Lys Thr Gly Phe 1 5 11 8 PRT Artificial Novel Sequence 11 Gly Asn Tyr Ser Gln Gln Leu Gln 1 5 12 4 PRT Artificial Novel Sequence 12 Leu Asp Asp Ser 1 13 5 PRT Artificial Novel Sequence 13 Leu Asp Asp Ser Ile 1 5 14 5 PRT Artificial Novel Sequence 14 Phe Leu Asp Asp Ser 1 5 15 6 PRT Artificial Novel Sequence 15 Leu Asp Asp Ser Ile Cys 1 5 16 7 PRT Artificial Novel Sequence 16 Phe Leu Asp Asp Ser Ile Cys 1 5 17 5 PRT Artificial Novel Sequence 17 Asp Asp Ser Ile Cys 1 5 18 8 PRT Artificial Novel Sequence 18 Tyr Met Met Gly Asn Glu Leu Thr 1 5 19 4 PRT Artificial Novel Sequence 19 Ser Ile Lys Ser 1 20 5 PRT Artificial Novel Sequence 20 Ser Ile Lys Ser Leu 1 5 21 5 PRT Artificial Novel Sequence 21 Val Ser Ile Lys Ser 1 5 22 6 PRT Artificial Novel Sequence 22 Ser Ile Lys Ser Leu Lys 1 5 23 7 PRT Artificial Novel Sequence 23 Val Ser Ile Lys Ser Leu Lys 1 5 24 5 PRT Artificial Novel Sequence 24 Ile Lys Ser Leu Lys 1 5 25 8 PRT Artificial Novel Sequence 25 Lys Thr Lys Gly Ser Gly Asn Thr 1 5 26 4 PRT Artificial Novel Sequence 26 Pro Gly Gln Asp 1 27 5 PRT Artificial Novel Sequence 27 Pro Gly Gln Asp Cys 1 5 28 5 PRT Artificial Novel Sequence 28 Gln Pro Gly Gln Asp 1 5 29 6 PRT Artificial Novel Sequence 29 Pro Gly Gln Asp Cys Arg 1 5 30 7 PRT Artificial Novel Sequence 30 Gln Pro Gly Gln Asp Cys Arg 1 5 31 5 PRT Artificial Novel Sequence 31 Gly Gln Asp Cys Arg 1 5 32 9 PRT Artificial Novel Sequence 32 Leu Ala Ala Phe Pro Glu Asp Arg Ser 1 5 33 321 DNA Artificial Novel Sequence 33 gatatccaga cgacacagac tacatcctcc cgttctgcct ctctgggaga cagagtcacc 60 atcagttgca gggcaggtca ggacattagt aattatttaa actggtatca gcagaaacca 120 gatggaactg ttaagctcct gatctactac acatcaagat tacactcagg agtcccatca 180 aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240 gaagatattg ccacttactt ttgccaacag ggtcatacgc ttccgtggac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 34 107 PRT Artificial Novel Sequence 34 Asp Ile Gln Thr Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Gly Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly His Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 35 363 DNA Artificial Novel Sequence 35 gatgtgcagc ttcaggagtc gggacctggc ctggtgaaac cttctcagtc tctgtccctc 60 acctgcactg tcactggcta ctcaatcacc agtgattatg cctggaactg gatccggcag 120 tttccaggaa acaaactgga gtggatgggc tacataagat acagtggtag tactagctac 180 aatccatctc tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc 240 ctgcagttga attctgtgac tactgaggac acagccacat attactgtgc aagagattgg 300 ccgcgaccga gctactggta cttcgatgtc tggggcgcag ggaccacggt caccgtctcc 360 tca 363 36 121 PRT Artificial Novel Sequence 36 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Arg Tyr Ser Gly Ser Thr Ser Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Asp Trp Pro Arg Pro Ser Tyr Trp Tyr Phe Asp Val Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 37 360 DNA Artificial Novel Sequence 37 caggtccaac tgcagcagtc cggacctgag ctggtgaagc cggggacttc agtgaggatt 60 tcctgcgagg cttctggcta caccttcaca agctactata tacactgggt gaaacagagg 120 cctggacagg gacttgagtg gattggatgt atttatcctg gaaatgtcaa tactaactat 180 aatgagaagt tcaaggacaa ggccacactg attgtagaca catcctccaa cactgcctac 240 atgcagctca gcagaatgac ctctgaggac tctgcggtct atttctgtac aagatcacac 300 tacggcctcg actggaactt cgatgtctgg ggcgcaggga ccacggtcac cgtctcctca 360 38 120 PRT Artificial Novel Sequence 38 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Thr 1 5 10 15 Ser Val Arg Ile Ser Cys Glu Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Cys Ile Tyr Pro Gly Asn Val Asn Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Ile Val Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Arg Met Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Thr Arg Ser His Tyr Gly Leu Asp Trp Asn Phe Asp Val Trp Gly Ala 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 39 321 DNA Artificial Novel Sequence 39 gacatccaga tgaaccagtc tccatccagt ctgtctgcat cccttggaga cacaattacc 60 atcacttgcc atgccagtca aaacatttat gtttggttaa actggtacca gcagaaacca 120 ggaaatattc ctaaactctt gatctataag gcttccaacc tgcacacagg cgtcccatca 180 aggtttagtg gcagtggatc tggaacaggc ttcacattaa ccatcagcag cctgcagcct 240 gaagacattg ccacttacta ctgtcaacag ggtcaaactt atccgtacac gttcggaggg 300 gggaccaagc tggaaataaa a 321 40 107 PRT Artificial Novel Sequence 40 Asp Ile Gln Met Asn Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Thr Ile Thr Ile Thr Cys His Ala Ser Gln Asn Ile Tyr Val Trp 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Asn Ile Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Asn Leu His Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gly Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Gln Thr Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 41 15 PRT Artificial Novel Sequence 41 Gly Asn Tyr Ser Gln Gln Leu Gln Val Tyr Ser Lys Thr Gly Phe 1 5 10 15 42 15 PRT Artificial Novel Sequence 42 Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser Ile Cys 1 5 10 15 43 15 PRT Artificial Novel Sequence 43 Lys Thr Lys Gly Ser Gly Asn Thr Val Ser Ile Lys Ser Leu Lys 1 5 10 15 44 16 PRT Artificial Novel Sequence 44 Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg 1 5 10 15 

1. A protein or peptide comprising the C′-D loop of a member of the CD28 family or containing a peptide analogous thereto or containing a mimicry compound thereto, not however a member of the CD28 family.
 2. A peptide according to claim 1, wherein the ends thereof are bound to one binding position each of a substrate, wherein the binding positions of the substrate are spatially arranged with regard to each other according to the binding positions for the C′-D loop in CD28, wherein the C′-D loop or the peptide being analogous thereto is fixed in a three-dimensional configuration according to the C′-D loop in CD28, the bound C′-D loop or the peptide being analogous thereto being freely accessible for antibodies, and wherein the substrate is not a member of the CD28 family without a C′-D loop or a natural peptide being analogous thereto of the respective CD28 family member.
 3. A peptide or protein according to claim 1 or 2, comprising an amino acid sequence seq.-ID 41, seq.-ID 1 or 5-10, not however human CD28, seq.-ID 42, seq.-ID 2 or 12-17, not however human CTLA-4, seq.-ID 43, seq.-ID 3 or 19-24, not however human ICOS, or seq.-ID 44, seq.-ID 4 or 26-31, not however human PD-1.
 4. A peptide or protein or mimicry compound according to one of claims 1 to 3, obtainable by that one or more prospective proteins or peptides are subjected to a binding test with one of the monoclonal antibodies (mAbs) 9D7 or 5.11A, and binding peptides or proteins are selected.
 5. A nucleic acid coding for a peptide according to one of claims 1 to 4 or for a protein containing this peptide, not however coding for a member of the CD28 family.
 6. A vector comprising a nucleic acid according to claim 5 operably linked to a suitable promotor for expression in a target cell line being transfected with the vector.
 7. Use of a peptide or protein or mimicry compound thereto according to one of claims 1 to 4 for producing a pharmaceutical composition for the modulation of the T cell proliferation.
 8. Use of a peptide according to one of claims 1 to 4 or of a protein containing this peptide or of a mimicry compound thereto, optionally lacking the binding site for costimulatory mAbs, in a method for producing mAbs which superagonistically modulate the proliferation of T cells of several to all sub-groups, wherein a non-human mammal is immunized with the protein or peptide or the mimicry compound thereto, wherein from the non-human mammal cells are taken, and hybridoma cells are produced from the cells, and wherein the thus obtained hybridoma cells are selected such that in their culture supernatant mAbs are contained which bind to the C′-D loop of the peptide or protein or to the mimicry compound thereto.
 9. Use of a peptide according to one of claims 1 to 4 or of a protein containing this peptide or of a mimicry compound thereto, optionally lacking the binding site for costimulatory mAbs, in a screening method for the identification of substances superagonistically modulating the proliferation of T cells of several to all sub-groups, wherein a prospective substance or a mixture of prospective substances is subjected to a binding assay with the peptide or protein or mimicry compound, and wherein substances binding to the peptide or protein or mimicry compound are selected, in particular mAbs and/or mimicry compounds.
 10. Hybridoma cells producing mAbs binding to a peptide or protein according to one of claims 1 to 4, in particular as filed under the DSM numbers DSM ACC2531 (9D7 or 9D7G3H11) or DSM ACC2530 (5.11A or 5.11A1C2H3).
 11. mAbs obtainable from hybridoma cells according to claim 10 or mAbs which are coded at least partially by one or more of the sequences seq.-ID 33, 35, 37 and/or 39, or mAbs which contain or consist of one or more sequences seq.-ID 34, 36, 38 and/or 40 or of FIG. 10 or sequences being homologous thereto.
 12. Use of a mAb according to claim 11 or of a mimicry compound thereto or of a substance obtainable from a screening method according to claim 9 for producing a pharmaceutical composition for the treatment of diseases with pathologically reduced CD4 T cell counts, in particular AIDS.
 13. Use of a mAb according to claim 11 or of a mimicry compound thereto or of a substance obtainable from a screening method according to claim 9 for producing a pharmaceutical composition for the treatment following stem cell transplantations after chemo or radio therapy of leukemic diseases.
 14. Use of a mAb according to claim 11 or of a mimicry compound thereto or of a substance obtainable from a screening method according to claim 9 for producing a pharmaceutical composition for multiplying and/or qualitatively influencing immune reactions after vaccinations.
 15. Use of a mAb according to claim 11 or of a mimicry compound thereto or of a substance obtainable from a screening method according to claim 9 for producing a pharmaceutical composition for the treatment of autoimmune-inflammatory diseases. 